�Vitex negundobelongs to the family Verbenaceae. It is a
large aromatic shrub distributed throughout the greater part of India up to
an altitude of 1500 m in the outer Himalayas. It is widely planted as a hedge
plant along the roads and between the roads. Traditionally it is having the
flok claims like useful in treatment of rheumatism, insecticidal,
antimicrobial, anticancer, tranquillizer, tonic, febrifuge, expectorant and
diuretic properties. In the present study an attempt had been made to
evaluate comparative antibacterial and antifungal principles from Vitex nigundo with some
therapeutically used antibiotics. Different extracts ofVitex negundo��leaves�
were investigated� for� its�
anti microbial� and antifungal activity
on� five bacterial species ��and� three
fungal species these are Staphylococcus
aureus, Proteus vulgaris, Bacillus subtilis, E.coli, Pseudomonas aerugenosa
and Aspergillus niger,� Aspergillus
flavon, Candida albicans respectively. Among all extracts water-ethanol
(50:50) extract showed maximum anti microbial and water extract showed
maximum antifungal activity against all tested species.�

��������� Vitex
negundobelonging to
family Verbenaceae is a large aromatic shrub distributed throughout the
greater part of India up to an altitude of 1500 m in the outer Himalayas[3]
It is a gregarious shrub that is found abundant along the banks or rivers, in
moist situations, open waste lands and near the deciduous forests. It is
widely planted as a hedge plant along the roads and between the roads. The
leaves are tri-or pentafoliate, lateral leaflets are smaller and nearly glabrous.
The shrub is one of the important plants used in Indian medicine. Almost all
parts of the herb are useful as a drug but the leaves and roots are most
important and sold as drugs[2].

����������������� Traditionally it is having
the flok claims like useful in treatment of rheumatism, insecticidal,
antimicrobial, anticancer, tranquillizer, tonic, febrifuge, expectorant and
diuretic properties.�

Material and Methods

Plant material

Fresh leaves ofVitex negundo collected in the
month of August to September from Amravati, Maharashtra and authenticated
from Prof. Dr.Marathe (Taxonomist), Botany Department, Vidharbha Institute of
Humanities and Science, Amravati, Maharashtra. The fresh leaves of Vitex negundowere
dried under shade & powdered in a mixer grinder. The powder leaves packed
in a paper bags & stored in air tight container until use.

Preparation
of extracts

The powdered leaves material was first defatted
with petroleum ether and further extracted with benzene, chloroform, ethanol,
water-ethanol (50:50) and water .Cold Maceration technique used for water extract.
Qualitative phytochemical analysis of all extracts was performed to know chemical constituents of extracts.[4-5]

Antimicrobial Study[6]

Microorganisms: All the strains of
micro- organism were obtained from National chemical Laboratory; Pune. Table
1 is showing Gram-Positive, Gram-negative and fungal microorganisms used for
the study.

Table 1: Gram-Positive, Gram-negative
and fungal microorganisms.

S. No.

Name of micro organism

CODE

1.

Staphylococcus aureus

NCIM 2079

2.

Proteus vulgaris

NCIM 2813

3.

Bacillus subtilis

NCIM 2063

4.

E.coli

NCIM 2065

5.

Pseudomonas aerugenosa

NCIM 2036

6

7.

Aspergillus niger

Aspergillus flavon

NCIM 545

NCIM-610

8.

Candida albicans

NCIM 3100

Antibacterial assay

Agar-well
diffusion method: The agar
diffusion method was used to screen the antibacterial activity of various
extract of leaves of.Vitex
negundo . 0.2 ml of each of the seeded broth containing 10-7 test organisms was inoculated on the
plates of solidified agar and spreaded uniformly. Then eight wells were cut
in the agar layer of each plate with an aluminum bore of 6mm diameter. In
every plate 6 different extracts of concentration 20 mg/ml dissolved in DMSO
were added while in 7th and 8th well standard
tetracycline and control DMSO was added. Then all plates were incubated at 370C
� 1 for 18 hrs. After the incubation period the mean diameter of the zone of
inhibition in mm obtained around the well was measured which has been shown
in Table� 2.

Antifungal assay:
Anti-fungal study was carried out through same procedure as used in
antibacterial study only difference was media used for antifungal study was
Sabouraud dextrose agar media (SDA MEDIUM).[6, 7�
]Results are shown in Table no.2.

Determination of Minimum Inhibitory
Concentration (MIC)

������
Two fold serial dilution method: The test was carried out individually for different
microorganisms with respect to the benzene, water-ethanol (50:50) extract
which shows comparative more effective result than other solvent on bacteria.
This testing was done in the seeded broth by two-fold serial dilution
technique. The solutions of different extracts were prepared of concentration
20 mg/ml in DMSO. For both Gram positive and Gram-negative organism a series
of 7 assay tubes for concentrations i.e. 20, 10, 5, 2.5, 1.25, 0.625, 0.3125
mg/ml for each extract were used. While standard tetracycline concentration
was taken as 5, 2.5, 0.625, 0.3125, 0.15625, 0.078072 mg/ml. One� positive�
control� for� each�
microorganism� was� prepared�
by� adding� nutrient�
broth� with� respective�
microorganism� inoculums.To each concentration test tube
required volume of sterile nutrient broth and inoculums was added and kept
for 24 hours incubation at 370C.�
After incubation period the growth of microorganism by considering
turbidity was measured by using turbidometer [8] .Results are shown
in Table 3.

Results and Discussion

����������
Phytochemical investigations shows that Vitex negundo
leaves contain essential oil and a component of the oil includes -pinene,
camphene, caryophyllene, citral, glycosides like negundoside, nishinadaside
and other hydroxybenzoylmussaenosidic acid derivatives. The flavonoids
reported from the leaves includes 5-hydroxy 3, 6, 7, 3�, 4�-pentamethoxy
flavone and 3, 5-dihydroxy-6, 7, 3�, 4�-tetramethoxyflavonal.A
new furanoerimophilane aldehyde have also been reported from leaves [1]

Aspergillus niger

5

2.5

5

Candida
albicans

10

10

10

Aspergillus
flavon

10

5

5

Conclusion

����� �AsVitex negundobelonging to family
Verbenaceae is already a well known herb for its anti-inflammatory activity
and from the above study we can also conclude that it also exhibits good antimicrobial and
antifungal activity against various bacterial and fungal strains.